In spite of numerous attempts to determine the roles and regulation of autolysins, their actual physiological function(s) and molecular mechanism of regulation remain elusive. Recently reported was the isolation and characterization of an autolytic-deficient mutant (called lyt-1) of S. aureus, which showed a single autolytic band instead of more than 10 autolytic bands in the parent strain. The major loss of autolytic activity without any effect on cell growth and development in the mutant strain suggests a redundant or nonessential role for the missing autolysins. lytic enzyme band present in the mutant may be vital for bacterial growth and lence in a rat model of experimental endocarditis, supporting a role for autolysins in pathogenicity. The objectives of this study are: (1) Molecular cloning and sequencing of the lytic gene (called lytM) still functional in the autolysis-defective lyt-1 mutant. This will be accomplished by constructing a genomic library of the mutant strain in E. coli and screening for lytic-positive clones using zymographic techniques. Deletion analysis in conjunction with lytic assay will be used to determine the minimum size of the insert required for the lytic activity. The dideoxynucleotide chain termination method will be used to determine the nucleotide sequence of the lytM gene. (2) Overexpression and purification, followed by biochemical characterization of the LytM enzyme. This objective will be accomplished by subcloning the coding sequence of lytM into a high level expression vector. The enzyme will be purified by either peptidoglycan affinity gels or HPLC. The biochemical properties of the enzyme will be determined with respect to substrate specificity, molecular mass, effect of temperature, pH, divalent cations, and other agents on enzyme activity. (3) Determination of LytM's biological role in S. aureus. This objective will be accomplished by using a site-directed mutagenesis technique to generate mutations in lytM followed by genetic studies. The mutated gene will be transferred into the parent and other lysis-defective mutant strains to determine their effects on bacterial growth and cell division of S. aureus.